1. Field of the Invention
This invention relates generally to the fields of biochemistry and cell biology and, more specifically, to a novel .beta.-1.fwdarw.6-N-acetylglucosaminyltransferase.
2. Background Information
The carbohydrate components of cell surface glycoproteins play important roles in a variety of biological processes, such as cell-cell interaction, cell adhesion, proliferation and differentiation. Specific sets of carbohydrates are characteristic for different stages of differentiation, and often these carbohydrates are recognized by specific antibodies, thus providing differentiation antigens. The aberrant expression of cell surface glycoproteins has been associated with various pathologies, including immunodeficiency syndromes and cancer.
Sialylated fucosylated lactosamines are a specific class of carbohydrates whose expression on the cell surface is regulated during normal development and pathogenesis. These sialylated fucosylated lactosamines are critical components of ligands of a family of cell adhesion receptors called selectins, which include E-, P- and L-selectins. Selectins normally play a role in mediating cell adhesion between leukocytes and the vascular endothelial surface, and therefore are critical in normal immune surveillance as well as in leukocyte recruitment to sites of acute and chronic inflammation. During metastasis of tumor cells, interactions between sialyl Le.sup.x -containing ligands and selectins appear to be important in modulating the adhesion of carcinoma cells to endothelial cells in target organs. For example, increased expression of sialyl Le.sup.x and sialyl Le.sup.a on the surface of carcinoma cells is positively associated with metastatic potential.
Sialyl Le.sup.x and sialyl Le.sup.a determinants are present in a variety of mucin-type glycoproteins that contain (O)-linked oligosaccharides, or O-glycans. Sialyl Le.sup.x, which has the structure NeuNAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.4(Fuc.alpha.1.fwdarw.3)GlcNac.bet a..fwdarw.R, and sialyl Le.sup.a, which has the structure NeuNAc.alpha.2.fwdarw.3Gal.beta.1.fwdarw.3(Fuc.alpha.1.fwdarw.4)GlcNac.bet a..fwdarw.R, are formed by modifications to oligosaccharides having the branched core 2 structure Gal.beta.1.fwdarw.3(GlcNAc.beta.1.fwdarw.6)GalNAc, as shown in FIG. 2, and also by modifications to oligosaccharides with linear or branched poly-N-acetyllactosamine repeats (Gal.beta.1.fwdarw.4GlcNac.beta.1.fwdarw.3), such as i or I antigen core structures.
As shown in FIG. 1, core 2 oligosaccharides are derived from core 1 structures by the action of a core 2 .beta.-1.fwdarw.6-N-acetylglucosaminyltransferase (C2GnT). A human enzyme with core 2 .beta.1.fwdarw.6 N-acetylglucosaminyltransferase activity, now designated C2GnT-L, has been purified and cloned and is the subject of U.S. Pat. No. 5,360,733. Recently, increased expression of C2GnT-L has been shown to be closely correlated with invasiveness of colorectal cancer cells, and ectopic expression of this enzyme in T cells is associated with an impaired immune response.
FIG. 3 shows the biosynthesis of the I branched antigen structure. The conversion of a linear poly-N-acetyllactosamine designated "i antigen" to the branched poly-N-acetyllactosamine designated "I antigen" is due to activity of an I-branching .beta.1.fwdarw.6 N-acetylglucosaminyltransferase (IGnT). A human I-branching .beta.1.fwdarw.6 N-acetylglucosaminyltransferase is the subject of U.S. Pat. No. 5,484,590. I branched poly-N-acetyllactosamines expressing blood group H antigens have much better avidity to anti-ABO antibodies than linear i poly-N-acetyllactosamines, and are implicated in detrimental immune responses when mother and fetus have incompatible blood group antigens. Additionally, more highly branched sialyl Le.sup.x -containing oligosaccharides have greatly increased affinity for selecting. Therefore, highly branched sialyl Le.sup.x -containing oligosaccharides could be administered as therapeutic agents that act as selectin ligand agonists or antagonists.
Core 4 oligosaccharides are derived from core 3 structures by the action of a core 4 .beta.-1.fwdarw.6-N-acetylglucosaminyltransferase (C4GnT), as shown in FIG. 1. Like core 2 and I-branched oligosaccharides, core 4-containing oligosaccharides are also important in normal development and in pathological conditions. For example, core 4 containing oligosaccharides are normally found in cells of the gastrointestinal tract. The levels of core 4 structures have been observed to be reduced in colon carcinoma cells.
These observations establish core 2, core 4 or I branching .beta.1.fwdarw.6 N-acetylglucosaminyltransferases and heir products as important mediators of normal development and differentiation, and additionally implicate these molecules in pathological conditions such as inflammation, tissue rejection and tumor metastasis. Identifying glycosyltransferases and their products is a critical step in preparing reagents that can be used to diagnose, prevent or treat these pathologies. Thus, a need exists to identify and characterize members of the glycosyltransferase enzyme family. The present invention satisfies this need and provides related advantages as well.